Antimicrobial peptides represent a potential alternative to conventional antibiotics, particularly against bacteria that have developed drug resistance. Although several previous studies have noted the antimicrobial properties of histone-derived antimicrobial peptides (HDAP), the structure-function relationships of these peptides are generally not well understood. The best characterized HDAP, buforin II (BF2), is an intriguing peptide that can translocate into cells, where it is thought to bind nucleic acids. The ability of BF2 to enter cells also gives it potential utility for drug delivery applications. Despite this knowledge about BF2, it is unknown whether other HDAPs share its membrane properties. This proposed research will involve three specific aims investigating HDAP function. First, the role of proline in the translocation and bacterial selectivity of BF2 will be investigated using a series of peptide variants. Since proline residues are common in antimicrobial peptides, including other HDAPs, these studies will provide general insights into the role played by proline in membrane-active peptides. The translocation and membrane interactions of variants will be characterized using a combination of lipid vesicle experiments, cellular assays and molecular dynamics simulations. These variants will also be used to investigate how proline residues promote BF2 preferentially targeting bacteria over eukaryotic cells. Second, a series of experiments will be performed to determine whether other known HDAPs, including parasin and hipposin, share membrane properties with BF2. To this end, this aim will use vesicle studies and cellular assays to measure the membrane permeabilization and translocation of peptides containing different combinations of parasin, BF2, and hipposin domains. Finally, a set of three novel HDAPs has been designed based on histone crystal structures and known characteristics of BF2. In the third aim, the membrane properties of these novel HDAPs will be evaluated using the experimental and computational methods applied in other parts of the proposal to characterize BF2 and other known HDAPs. Overall, these studies will provide insights into what membrane properties are shared within the HDAP family. Moreover, this information will serve as a foundation for using histones as a framework for designing novel antimicrobial and cell-penetrating peptides with desired properties. PUBLIC HEALTH RELEVANCE: Antimicrobial peptides, which are small proteins produced by a wide variety of organisms, represent a potential alternative to combat bacteria that are resistant to conventional antibiotics. This proposed research will provide detailed information about how a particularly intriguing family of antimicrobial peptides, the histone-derived antimicrobial peptides, functions. Improving our understanding of these peptides will help researchers develop novel peptides for therapeutic use.